Effects of gamete behavior and density on fertilization success in marine green algae: insights from three-dimensional numerical simulations

We developed a numerical simulation of mating experiment to study effects of phototactic gamete behavior and density on fertilization success, using the C++ programming language, and pseudo-parallelization methods with input parameters based on experimental data. In our experiments, we found that gametes with positive phototaxis are favored, particularly in shallow water, because they can search for potential mates on the two-dimensional (2-D) water surface rather than randomly in three dimensions. We also found evidence that sperm (male gametes) limitation might not be the dominant selective force in the evolution of isogamous or slightly anisogamous marine green algae because almost all of female gametes can be fertilized on the 2-D water surface meaning they might not be under sperm limited conditions. Gamete density also appears to affect mating success seriously. These findings were produced by some technical progress made recently to rapidly and correctly count the numbers of zygotes formed calculating the locations of huge numbers of male and female gametes in the test tank. Both gamete behavior and density might be determined by environmental conditions of habitat, particularly the depth of water.     

Gametic conflict versus contact in the evolution of anisogamy

Anisogamy refers to gametes that differ in size, and characterizes the difference between males and females. The evolution of aniosgamy is widely interpreted as involving conflict between gamete producers with small sperm parasitizing on the investment made by the eggs. Using a population genetic model for evolution at a locus that codes jointly for sperm and egg sizes of a hermaphrodite, we show that the origin of anisogamy in an externally spawning population need not involve conflict between gamete producers. Gamete size dimorphism may be an adaptation that increases gamete encounter rates when large zygotes are selected, and we show this in a mechanistically general individual selection model. We use the Vance survival function without specific allometric assumptions to model the zygote fitness dependence on its size, and hence obtain ecological and life-history correlates of isogamy and anisogamy, which we successfully compare with data from Volvocales.     

Selection for high gamete encounter rates explains the evolution of anisogamy using plausible assumptions about size relationships of swimming speed and duration

A previous general model describing physical constraints on gamete encounter rates was modified to incorporate assumptions that increased size causes decreased swimming speed and increased fertile period (or other proportional enhancement to gamete fertility). The analysis indicates that with moderately strong size dependence of fertile period and a range of speed dependencies, selection for high encounter rates pressures mating systems that develop any heritable difference in size between the gametes of different mating types to exaggerate the difference and evolve from isogamy to anisogamy. The smaller gamete has an optimal size, but the larger faces continuing selection for increased size. This continues to a size that is estimated to be sufficient to make pheromone production of sperm attractants practical. This mechanism then bridges the missing link between isogametes and oogamy in a previous analysis of the effectiveness of pheromones in explaining the success of male-female mating systems. The evolution and success of anisogamy and oogamy can be explained solely on the basis of physical effects on the encounter process.     

Evolution of gamete size in primitive taxa without mating types

An ESS model to better understand the evolutionary dynamics of a primitive non-mating type gamete size was developed with reference to the PBS (Parker, Baker and Smith’s) theory, which was based on total numbers of zygotes formed and the zygote survival rates. We did not include mating types since it has been suggested that primitive mating systems did not have mating types. As input parameters, we used experimental data on gamete motility of marine green algae. Based on hard sphere collision mechanics, we detailed the fertilization kinetics of gametes that swim in water prior to fusing with their partners through a set of coupled, non-linear differential equations. These equations were integrated numerically using typical values of the constant parameters. To estimate the relative zygote survival rate, we used a function that is sigmoid in shape and examined some evolutionarily stable strategies in mating systems that depend on optimizing values of the invasion success ratio.     

In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.     

Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.     

雄性川金丝猴对雌性的受孕认知与等级识别

社会认知是群居动物适应复杂环境和社会生活能力的体现,致力于理解自身、他人与社会。认知有利于促进利益分配的优化,而行为认知策略则是关注的热点问题。但迄今为止,川金丝猴如何进行社会地位与受孕状态等基础认知尚不清楚。本研究通过投食招引,在个体识别和等级判定的基础上,以栖息于秦岭观音山自然保护区熊猫谷景区的一个半野生川金丝猴群为对象,采用瞬时扫描和焦点动物取样法,观察并收集了猴群中6个一雄多雌单元成年雌雄个体的空间位置、邀配、交配及产仔行为数据,结合食物密度梯度"同心圆"和受孕周期,推测雌性成功受孕的交配时间段,运用配对t-检验统计证实受孕前后雌性的交配实现率差异显著(t=4.527,P=0.001),表现为受孕前雌性邀配行为获取的雄性响应率明显高于受孕后;通过Spearman相关性检验发现,雌性受孕前的交配实现率(R=0.527,P=0.006)及受孕时间(R=0.556,P=0.049)与自身等级均显著正相关,即高等级雌性邀配行为的雄性响应率及与雄性生殖性交配的时间明显高于或早于低等级雌性。结果表明,雄性川金丝猴拥有认知单元内成年雌性受孕状况和等级地位的能力,这种认知能力影响着雄性的性行为方式。     

Egg size in relation to fertilization dynamics in free-spawning tropical reef fishes

In marine invertebrates that spawn by simply releasing their gametes into the water (free-spawning), fertilization success likely is often limited by low sperm concentrations, due to dispersion of mates and dispersal of gametes by water movements. Production of large, low density eggs might be advantageous when sperm concentrations consistently are low, because large target size might increase egg/sperm encounters, and more low than high density eggs could be produced per clutch. Although average fertilization success in the labrid Thalassoma bifasciatum is 95% in both group spawns (in which multiple males compete for fertilizations by producing large quantities of sperm) and pair (mono-male) spawns, it is slightly lower in pair spawns, due to low level sperm limitation that arises because pair-spawning males release near the minimum number of sperm necessary for maximum fertilization. I examined whether variation in egg size and content in T. bifasciatum and other free-spawning fishes is related to variation in spawning mode, to assess whether compensatory production of large, low-density eggs might be contributing to high fertilization success in pair spawns. I found no difference between the volume or density of eggs of (1) pair- and group-spawning females of T. bifasciatum, or (2) pair-and group-spawning congeneric species of labrids, scarids, and serranids, or (3) labrids and scarids with vigorous, rapid spawning movements (which could turbulently diffuse gamete clouds) and those with slow movements. Further, egg density does not decline with increasing egg volume among those fishes. Assuming that egg size can affect fertilization success, then sperm limitation seems unlikely to represent a significant problem for pair-spawning T. bifasciatum, probably because mates place their vents close together during gamete release. The situation regarding sperm limitation in other fishes, and effects of environmentally generated water turbulence on it, are less clear. Interspecific variation in the size and content of these fishes' eggs may relate to provisioning of offspring for different larval life-histories.     

Attraction and transport of male gametes for fertilization

 Two capabilities are critical in attracting and transporting male gametes for fertilization: (1) the pollen tube must locate, enter and discharge its contents at the correct site within the female gametophyte, and (2) once inside the embryo sac, the non-motile male gametes must be transported to the egg and the central cells for double fertilization. This review summarizes current information about evidence for communication between embryo sac and pollen tube and the means by which the non-motile male gametes are transported from the aperture of the pollen tube to the site of gamete fusion. Received: 6 June 1996 / Revision accepted: 9 July 1996     

Simulation of gamete behaviors and the evolution of anisogamy: reproductive strategies of marine green algae

In marine green algae, isogamous or slightly anisogamous species are taxonomically widespread. They produce positively phototactic gametes in both sexes. We developed a new numerical simulator of gamete behavior using C++ and pseudo-parallelization methods to elucidate potential advantages of phototaxis. Input parameters were set based on experimental data. Each gamete swimming in a virtual rectangular test tank was tracked and the distances between the centers of nearby male and female were measured at each step to detect collisions. Our results shed light on the roles of gamete behavior and the mechanisms of the evolution of anisogamy and more derived forms of sexual dimorphism. We demonstrated that not only gametes with positive phototaxis were favored over those without, particularly in shallow water. This was because they could search for potential mates on the 2-D water surface rather than randomly in three dimensions. Also, phototactic behavior clarified the difference between isogamy and slight anisogamy. Isogamous species produced more zygotes than slightly anisogamous ones only under the phototactic conditions. Our results suggested that sperm limitation might be easily resolved particularly in the slightly anisogamous species. Some more markedly anisogamous species produce the smaller male gametes without any phototactic devices and the larger positively phototactic female gametes. In such species, female gametes attract their partners using a sexual pheromone. This pheromonal attraction system might have played a key role in the evolution of anisogamy, because it could enable markedly anisogamous species achieve 2-D search efficiencies on the water surface. The mating systems appear to be tightly tuned o the environmental conditions of their habitats.     

Localization of plasmidlike DNA in giant-celled marine green algae

Summary Novel extrachromosomal DNA molecules were localized in giant-celled marine green algae by organelle isolation and fluorescence in situ hybridization methodologies. Nucleic acids extracted from isolated chloroplasts ofErnodesmis verticillata andVentricaria ventricosa were greatly enriched in plasmidlike DNA species. Fluorescence in situ hybridization was employed to resolve further the subcellular location of these molecules. Cloned restriction fragments of the algal plasmidlike DNA hybridized solely to low-molecular-weight DNA in Southern blots; they did not hybridize to any chromosomal DNA. Probes were generated from these clones that either did (Northern-positive) or did not (Northern-negative) hybridize to RNA species in Northern blots. Probes specific for localizing the plasmidlike DNA were generated from the latter clones, whereas probes potentially localizing both DNA and relevant mRNA species were generated from the former ones. After hybridization and signal amplification via indirect immunofluorescence, fluorescent punctae were visible surrounding the single pyrenoid in each chloroplast with both types of probes. The punctae were arranged in a hollow spherical configuration, as resolved by confocal laser scanning microscopy. Nearly twice as many punctae per chloroplast were present inV. ventricosa (11.5) as there were inE. verticillata (6.0). The differential distribution of plasmidlike DNA within each chloroplast was in contrast to chloroplast chromosomal DNA, which occurred as multiple nucleoids scattered throughout the entire organelle. The localization of plasmidlike DNA within chloroplasts correlates well with previous sequence data indicating that these molecules contain putative open reading frames encoding protein components of photosystems I and II.Abbreviations CLSM confocal laser scanning microscopy - DAPI 4,6-diamidino-2-phenylmdole - FITC fluorescein isothiocyanate - FISH fluorescence in situ hybridization - HMW high molecular weight - LMW low molecular weight - ORF open reading frame     

Gametic behavior in a marine green alga, Monostroma angicava: an effect of phototaxis on mating efficiency

The role of phototactic behavior of gametes was tested experimentally in the slightly anisogamous marine green alga Monostroma angicava Kjellman, and the effect of phototaxis on mating efficiency was discovered. Both male and female gametes showed positive phototaxis in response to a white light source. In contrast, they did not respond to a red light source. Their swimming velocity did not differ between these two illuminating light sources. It was, therefore, suggested that the search ability of the gamete itself might not vary between phototactic and non-phototactic conditions. The number of zygotes formed during the mating process may be expressed as the product of the number of encounters between male and female gametes and the fraction of encounters that result in sexual fusion. In this study, with high densities of male and female gametes mixed in test tubes, almost all minor (fewer in number) gametes fused sexually within 10 min. After dilution of the gamete suspensions by half, mating efficiency in test tubes illuminated by white light from above was higher than that in dark controls. This suggests that male and female gametes gathered at the water surface through their positive phototaxis, thus increasing the rate of encounters. Mating efficiency also decreased if the test tubes were illuminated from above by white light and also shaken. Since negative phototaxis is clearly shown in planozygotes, we suggest that positive phototaxis of male and female gametes in M. angicava is an adaptive trait for increasing the rate of gametic encounters rather than for the dispersal of zygotes as previously reported for zoospores of some marine algae. Received: 12 February 1999 / Revision accepted: 24 May 1999     

The kinetics of monospermic and polyspermic fertilization in free-spawning marine invertebrates

The equation of Vogel et al. (1982) is widely used in fertilization studies of free-spawning marine invertebrates to predict the percentage of viable eggs that will be fertilized at any specified levels of gamete concentration and contact time. Here, the random collision model that underlies the Vogel et al. equation is extended to distinguish between monospermic and polyspermic fertilization, and separate equations for the percentages of monospermic and polyspermic fertilization are obtained. These equations provide an explanation for empirical observations which have shown a decreased percentage of successful egg development at high sperm concentrations. Comparison is made with an earlier heuristic attempt (Styan, 1998) to predict the extent of polyspermic fertilization, and it is found that this earlier method can underestimate the percentage of polyspermic fertilization by up to 10 percent. Moreover, the approach used here retains the flexibility to model changes in sperm concentration due to dispersal mechanisms, and is able to model different mechanisms for the block to polyspermy.     

The role of oocyte maturation in the ontogeny of the fertilization site in the hydrozoan Hydractinia echinata

Summary In hydrozoans the sperm will fuse with the egg only at the site of polar body formation. The primary oocyte and maturing oocytes which have produced the first polar body cannot be fertilized even though maturing oocytes which have produced the first polar body attract sperm. These eggs do not acquire the ability to be fertilized until after second polar body formation. If either first or second polar body formation is inhibited or if first and second polar body formation do not take place in close proximity to each other, the fertilization site is not set up. Under normal circumstances the site of polar body formation takes place at the region on the maturing oocyte surface nearest the site where the germinal vesicle resided in the primary oocyte. When maturing oocytes are centrifuged prior to polar body formation, the site of polar body formation is frequently shifted so that it does not correspond to the site where it would be given off under normal circumstances. Under these conditions the shifted site of polar body formation is the only site where the egg can be fertilized, indicating that the fertilization site is selected during oocyte maturation.Oocyte maturation in these hydrozoans is mediated by a hormone released by the somatic cells of gonophores as a consequence of bringing dark adapted gonophores into the light. The hormone acts directly on the oocyte to induce maturation. The oocyte only has to be exposed to the hormone for the first few minutes of the maturation process in order to complete the process of maturation.Dedicated to Professor N.H. Verdonk of the Rijksuniversiteit Utrecht on his 65th birthday     

Effect of external pH on inorganic carbon assimilation in unicellular marine green algae

Carbonic anhydrase (CA) induction has been studied in three marine green algae under acidic (pH 4.5) or alkaline (pH 8.0) conditions. An inhibition of the induction of the external CA in acidic conditions, similar to that observed in some freshwater green algae, could be observed in only Chlorella saccharophila. In the two other species, Chlorococcum littorale and Stichococcus bacillaris, no significant difference in CA induction was found under two pH conditions. The exact function of the external CA of C. saccharophila remains unclear, since cells grown under acidic conditions (under which this enzyme is repressed) possess the same abilities to use inorganic carbon (Ci) as alkaline‐grown cells. Internal pH values were not modified by the pH of the medium used to cultivate C. saccharophila. Regardless of the growth conditions, activities related to carbon fixation, that is, photosynthetic oxygen evolution, Ci uptake and assimilation were enhanced when the measurements were performed at acidic pH. This indicates that this marine alga is able to use CO₂ more efficiently than HCO₃^?. No evidence could be found for a specific Ci uptake and assimilation system in the acid‐grown cells.     

Successive observations on the fertilization of a centric diatomMelosira moniliformis var.octagona

The details of fertilization in the centric diatom,Melosira moniliformis var.octagona, were examined by light microscopy with a video system. The sperm penetrated into the egg cell through a temporary opening of the oogonium. The flagellum of the sperm was brought into the egg cell and it remained active for a few minutes. Three observations on the behavior of the sperm and egg cell during fertilization are shown.     

Repeated evolution and reversibility of self‐fertilization in the volvocine green algae*

Outcrossing and self‐fertilization are fundamental strategies of sexual reproduction, each with different evolutionary costs and benefits. Self‐fertilization is thought to be an evolutionary “dead‐end” strategy, beneficial in the short term but costly in the long term, resulting in self‐fertilizing species that occupy only the tips of phylogenetic trees. Here, we use volvocine green algae to investigate the evolution of self‐fertilization. We use ancestral‐state reconstructions to show that self‐fertilization has repeatedly evolved from outcrossing ancestors and that multiple reversals from selfing to outcrossing have occurred. We use three phylogenetic metrics to show that self‐fertilization is not restricted to the tips of the phylogenetic tree, a finding inconsistent with the view of self‐fertilization as a dead‐end strategy. We also find no evidence for higher extinction rates or lower speciation rates in selfing lineages. We find that self‐fertilizing species have significantly larger colonies than outcrossing species, suggesting the benefits of selfing may counteract the costs of increased size. We speculate that our macroevolutionary results on self‐fertilization (i.e., non‐tippy distribution, no decreased diversification rates) may be explained by the haploid‐dominant life cycle that occurs in volvocine algae, which may alter the costs and benefits of selfing.     

The eyespot of the flagellateTetraselmis cordiformis stein (Chlorophyceae): Structural spezialization of the outer chloroplast membrane and its possible significance in phototaxis of green algae

Summary The eyespot region of the flagellateTetraselmis cordiformis Stein (Chlorophyceae) was investigated with the freeze-fracture technique. The only fracture faces observed in this region were the two complementary fracture faces (PF and EF) of the outer chloroplast envelope membrane. Intramembranous particle numbers on both fracture faces of this membrane were significantly higher in the eyespot region as compared to regions outside the eye-spot. Higher numbers of particles on the PF face in the eyespot region were mainly caused by an increase in particle numbers of the size class 6–8 mm, while on the EF face particle size distribution was not significantly different between eyespot and other regions. Functional implications are discussed and evidence is presented that the outer chloroplast envelope membrane may be the site of photoreceptor location in green algal phototaxis.     

Induction of sexual behavior in male fish (Rhodeus ocellatus ocellatus) by amino acids

Summary A new function of amino acid in fish behavior was found. Amino acids induced sexual behavior in male rose bitterling (Rhodeus ocellatus ocellatus). Two types of sexual behavior which were pecking and sperm release were observed. Amino acids are known as feeding stimulants in some fish. The pecking behavior of male fish induced by amino acids is similar to the feeding behavior but it was sexual. Only male bitterling showed pecking and sperm release but the female showed no response to the amino acids. 10 out of 20 amino acids induced sexual behavior and both pecking and sperm release were induced by the same amino acids. These two kinds of behavior changed alternately depending on the light conditions. It is of interest that non-specific material such as some amino acids function like sex pheromone.